Microorganism capable of degrading phenolics

ABSTRACT

Mutant microorganism, Pseudomonas putida CB-173 degrading phenolics, and at a temperature as low as, e.g., about 1° to 4° C., at a faster rate than known Pseudomonas putida type strains, and process for treating wastewater containing phenolics using the mutant microorganism strain Pseudomonas putida CB-173.

This is a continuation of application Ser. No. 229,025, filed Jan. 27,1981, now U.S. Pat. No. 4,352,886.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a novel microorganism of the strainPseudomonas putida which has an enhanced ability to degrade phenolics inindustrial wastewater lagoons even at low temperature under aerobicconditions and to a process for treating wastewater to remove phenolicstherefrom utilizing such microorganism strain.

2. Description of the Prior Art

Many producers and processing plants which use large amounts of waterrender this water unsuitable for reuse and undesirable for release intothe biosphere due to the pollution problems which result when it isdischarged untreated. Biological systems, such as aerated lagoons andactivated sludge systems, are the generally accepted methods ofprocessing these wastewaters prior to reuse or discharge to receivingbodies of water.

While biological processes occurring during such a biological treatmentprovide the ability, with most wastewaters, to produce effluent whichhas both low biological oxygen demand (BOD), low chemical oxygen demand(COD), and low total suspended solids (TSS), unfortunately,conventionally employed biological treatment systems depend on atemperature range favorable to biological activity. Available literatureon biological treatment suggests that biological activity virtuallyceases below about 8° C. Large sums of money are required to heatlagoons in the winter months in many parts of the country to achievelagoon temperatures at which biological activity can proceed.

Often, lagoon efficiency suffers due to concentration build up duringwinter months when microorganisms are less active. In some cases,because of reduced lagoon efficiency the rate of discharge of effluentsinto the lagoons must be decreased during winter months.

With increased concern as to minimization of the problems arising frompollution, biological processes are being employed in industry in anincreasing amount, and a large amount of activity in research anddevelopment is occurring presently to develop new microbial strainscapable of use in wastewater treatment industrially, municipally anddomestically. Even with this increased activity in investigating anddeveloping strains of microorganisms to solve particular waste removalproblems, treatment at low temperatures still remains a problem.

SUMMARY OF THE INVENTION

Accordingly, an object of this invention is to provide a microorganismwhich is effective in degrading phenolics in wastewater at lowtemperatures.

A further object of this invention is to provide a treatment forindustrial, municipal and domestic wastewaters utilizing a novel mutantof Pseudomonas putida, alone or in combination with othermicroorganisms, to degrade and remove phenolics from wastewaters.

Another object of this invention is to provide a biological process forremoving phenolics from wastewater using a novel mutant strain ofPseudomonas putida in conjunction with a specific combination of othermicroorganisms described hereinafter, particularly at low temperatures.

An even further object of this invention is to provide a biologicaltreatment process effective even at low temperatures for treatingindustrial, municipal and domestic wastewaters containing phenolics andrendering such wastewaters suitable for discharge into the biosphere,thereby minimizing problems of pollution.

An additional object of this invention is to provide a novel mutantstrain of Pseudomonas putida.

In one embodiment of this invention, this invention provides a processfor treating wastewater containing phenolics, which comprises treatingwastewater containing phenolics with a microorganism of the strainPseudomonas putida CB-173.

In another embodiment of this invention, this invention provides a novelstrain of the species Pseudomonas putida CB-173 having thecharacteristics described below.

DETAILED DESCRIPTION OF THE INVENTION

The novel mutant Pseudomonas putida CB-173 (hereinafter "mutant strain")was produced by mutation of a parent strain of Pseudomonas putidaisolated from a wastewater lagoon at a large textile chemicalmanufacturing plant located in Wellford, S.C.

This novel mutant has been found to be capable of degrading phenolics attemperatures as low as 4° C. and lower at a rate faster than the typestrains of Pseudomonas putida, both biotype A and biotype B, and has thecharacteristics described below.

The mutant strain Pseudomonas putida CB-173 is a gram negative,non-spore forming rod. The cells are rod shaped, sometimes curved whichhave one to four polar flagella and the cells are motile. Approximately10-20% of the cells are joined in filaments of two cells each.

The mutant strain is a strict aerobe which produces a diffusibleyellow-green fluorescent pigment. On metabolism in the presence ofnitrate, the mutant strain does not produce nitrate reductase. Themutant strain is "catalase positive" and weakly positive for oxidase.There is no gelatin liquefaction by the mutant strain in eight days. Themutant strain utilizes various amines for growth, but does not use thealcohol m-inositol. A suitable growth temperature range is from about 1°C. to about 35° C. with optimal growth occurring at 25°-30° C. No growthis observed in two days at 37° C.

Other cultural characteristics and colonial morphology of this mutantstrain are shown in Tables 1-6 below.

In the following tables, Pseudomonas putida strains, biotype A (ATCC12633) and biotype B (ATCC 17536), were employed as known type strainsfor characterization purposes.

                  TABLE 1                                                         ______________________________________                                        MICROSCOPIC MORPHOLOGY                                                        CHARAC-   STRAIN                                                              TERISTIC  Biotype A** Biotype B**                                                                              CB-173                                       ______________________________________                                        Cell Size*                                                                    Length    2.0-4.0 μm                                                                             2.0-4.0 μm                                                                            2.0-4.0 μm                                Width     0.7-1.1 μm                                                                             0.7-1.1 μm                                                                            0.7-1.1 μm                                Gram Reaction                                                                           Negative Rod                                                                              Negative Rod                                                                             Negative Rod                                 ______________________________________                                         *Wet mounts of 20hour cultures viewed under phase contrast (1000×).     Sizes given in micrometers.                                                   **Data from Bergey's Manual of Determinative Bacteriology, 8th Ed., The       Williams and Wilkins Co., Baltimore (1974).                              

                  TABLE 2                                                         ______________________________________                                        COLONIAL CHARACTERISTICS OF PSEUDOMONAS                                       PUTIDA CB-173 AFTER 24 HOURS AT 30° C.                                 ______________________________________                                        Nutrient Agar                                                                            Circular, convex colonies with smooth, shiny                                  surface and entire edge. Colonies, 1.25-                                      1.75 mm in size, are transluscent with slight                                 cream color and produce a greenish-yellow                                     fluorescent pigment                                                Hecktoen Enteric                                                                         Circular, convex colonies with                                     Agar       smooth, shiny surface and entire edge.                                        Colonies are transluscent to opaque with                                      green color; 0.6-0.75 mm in diameter. No                                      pigment is produced.                                               Plate Count Agar                                                                         Circular, convex colonies with smooth,                                        shiny surface and entire edge. Colonies,                                      1.25-1.5 mm in diameter, are transluscent                                     with slight cream color and produce a                                         greenish-yellow fluorescent pigment.                               Trypticase Soy                                                                           Circular, convex colonies with smooth,                             Agar       shiny surface and entire edge. Colonies are                                   transparent with slight cream color and                                       produce a greenish-yellow fluorescent pig-                                    ment. Colonies are 1.25-2 mm in diameter.                          ______________________________________                                         NOTE: Plate Count Agar and Hecktoen Enteric Agar are products of Difco        Laboratories. Nutrient Agar and Trypticase Soy Agar are products of           Baltimore Biological Laboratories.                                       

                  TABLE 3                                                         ______________________________________                                        UTILIZATION OF CARBON-CONTAINING                                              COMPOUNDS FOR GROWTH                                                                     GROWTH RESPONSE**                                                  COMPOUND*    Biotype A   Biotype B CB173                                      ______________________________________                                        Carbohydrates                                                                 (& sugar de-                                                                  rivatives)                                                                    α-Cellulose                                                                          -           -         -                                          L-Arabinose  -           -         -                                          D-Trehalose  -           -         -                                          D-Cellobiose -           -         -                                          Xylose       +           -         -                                          D-Galactose  -           +         -                                          D-Mannose    -           +         -                                          D-Lactose    -           -         -                                          Alcohols                                                                      meso-Inositol                                                                              -           -         -                                          1-Butanol    +           +         +                                          1-Propanol   +           +         +                                          Aliphatic                                                                     Amino Acids                                                                   L-Alanine    +           +         +                                          β-Alanine                                                                             +           +         +                                          D-Alanine    +           +         +                                          L-Arginine   +           +         +                                          L-Valine     +           +         -                                          L-Asparagine +           +         +                                          L-Cystine    +           ±      ±                                       L-Leucine    +           +         -                                          Glycine      +           +         -                                          Pyridine     -           -         -                                          Cytosine     ±        -         -                                          D-Aspartic Acid                                                                            +           ±      ±                                       Amino Acids and                                                               Related Compounds                                                             Containing A Ring                                                             Structure                                                                     D-Histidine  +           ±      +                                          L-Proline    +           +         +                                          L-Tyrosine   +           +         +                                          Tryptophan   -           +         -                                          Fatty Acids                                                                   Heptanoic Acid                                                                             +           +         -                                          Dicarboxylic                                                                  Acids                                                                         Malonic      +           +         -                                          Succinic     +           ±      -                                          Saccharic    +           +         ±                                       Maleic       +           +         ±                                       Oxalic       +           ±      ±                                       Hydroxy Acids                                                                 Malic        +           +         +                                          D-Glyceric   -           -         -                                          Miscellaneous                                                                 Acids                                                                         Pyruvic      +           +         +                                          Citric       +           +         +                                          Nicotinic    +           -         -                                          Miscellaneous                                                                 Compounds                                                                     Igepal***    -           ±      +                                          CO 660                                                                        Igepal***    -           -         -                                          CO 520                                                                        EDTA         -           -         -                                          m-Cresol (100 mg/l)                                                                        -           -         -                                          Triacetin    -           -         -                                          ______________________________________                                         *Compound added at 0.5% to minimal salt medium (Roy Curtiss, III, J.          Bact., 89, pages 28-40 (1965)).                                               **+ indicates growth greater than that of blank;                              - indicates growth less than that of blank;                                   ± indicates growth approximately equal to blank or weak growth, after      seven days at 30° C.                                                   ***Trade name for a nonionic nonyl phenolethylene oxide condensate            produced by GAF.                                                         

                  TABLE 4                                                         ______________________________________                                        UTILIZATION OF NITROGENOUS COMPOUNDS                                          AS SOLE NITROGEN SOURCE                                                                    GROWTH RESPONSE**                                                COMPOUND*      Biotype A Biotype B   CB-173                                   ______________________________________                                        NH.sub.4 Cl    +         +           +                                        KNO.sub.3      +         +           -                                        D-Alanine      +         +           +                                        L-Aspartic Acid                                                                              -         -           -                                        L-Tyrosine     +         +           +                                        Betaine        +         +           +                                        Tryptophan     ±      +           +                                        No Nitrogen Compound                                                                         -         -           -                                        ______________________________________                                         NOTE: Fluorescent yellowgreen pigment produced on betaine by Biotype A        only, not by Biotype B and CB173.                                             *Nitrogenous compound added at 0.5% to minimal salts medium (Curtiss          (1965)), but without NH.sub.4 Cl and NH.sub.4 NO.sub.3, and agar, but wit     0.5 g Na Citrate/100 ml as a carbon source.                                   **+ indicates growth greater than that of blank;                              - indicates growth less than that of blank;                                   ± indicates growth approximately equal to blank or weak growth after       two days at room temperature in tubes aerated on a shaker.               

                  TABLE 5                                                         ______________________________________                                        CULTURE GROWTH IN PRESENCE OF HEAVY METALS                                    HEAVY     CONCEN-   STRAIN RESPONSE                                           METAL*    TRATION   Biotype A Biotype B                                                                              CB-173                                 ______________________________________                                        CdCl.sub.2                                                                              200    mg/l   +       +        -                                              20     mg/l   +       +        ±                                           2      mg/l   +       +        ±                                 CoCl.sub.2                                                                              200    mg/l   -       -        -                                              20     mg/l   +       +        +                                              2      mg/l   +       +        +                                    Na.sub.2 HAsO.sub.4                                                                     200    mg/l   +       +        +                                              20     mg/l   +       +        +                                              2      mg/l   +       +        +                                    HgCl.sub.2                                                                              400    mg/l   -       -        -                                              200    mg/l   -       -        -                                              20     mg/l   -       -        -                                              2      mg/l   +       +        -                                    Control                                                                       No heavy metal          +       +        +                                    ______________________________________                                         *Heavy metal added to minimal salts medium containing (0.5%) Dglucose         (Curtiss (1965)). CB173 does not grow as readily in this medium as Biotyp     A and Biotype B. Growth was scored by comparison with the control.            + indicates growth (no inhibition);                                           - indicates no growth (inhibition); and                                       ± indicates weak growth.                                              

                  TABLE 6                                                         ______________________________________                                        RESISTANCE TO ANTIBIOTICS                                                                 STRAIN GROWTH RESPONSE                                                          Biotype   Biotype                                               ANTIBIOTIC    A         B           CB-173                                    ______________________________________                                        Amakacin      S         S           S                                         Ampicillin    R         R           R                                         Carbenicillin R         R           R                                         Cefamandol    R         R           R                                         Cephalothin   R         R           R                                         Chloramphenicol                                                                             R         R           I                                         Gentamycin    S         S           S                                         Kanamycin     S         S           S                                         Streptomycin  I         S           S                                         Tetracycline  I         I           I                                         Tobramycin    S         S           S                                         ______________________________________                                         Growth response on Pfizer Antimicrobial Susceptibility Disks; Pfizer,         Inc.; Scored:                                                                 S = sensitive to antibiotic;                                                  R = resistant to antibiotic;                                                  I = intermediate.                                                        

On the basis of the morphological, cultural, and physiologicalcharacteristics set forth above, the mutant strain has been identifiedas a member of the species, Pseudomonas putida and has been designedherein as Pseudomonas putida CB-173.

A culture of the mutant strain has been deposited in the American TypeCulture Collection and has receiving an accession number, ATCC-31800.

As indicated above, the parent strain from which the mutant strain wasdeveloped was isolated from a wastewater lagoon of a textile chemicalmanufacturing plant in Welford, S.C. This wastewater was streaked onto anutrient supplemented sole source of carbon agar (NS-SSC). Thecomposition of the media used is set forth in Table 7 below:

                  TABLE 7                                                         ______________________________________                                        COMPOSITION OF NUTRIENT-SUPPLEMENTED                                          SOLE SOURCE OF CARBON AGAR                                                    Compound             Quantity                                                 ______________________________________                                        Nutrient Broth,* prepared                                                                          20        ml/l                                           (NH.sub.4).sub.2 SO.sub.4                                                                          2         g/l                                            Na.sub.2 HPO.sub.4   1         g/l                                            Phenol-liquefied, approx. 90%                                                                      1.05      ml/l                                           ______________________________________                                         Tap water used to provide trace minerals                                      *Product of Baltimore Biological Laboratories                            

After four days incubation at 4° C., growth appeared. The predominantorganism was mutated by exposure to ethyl methane sulfonate (EMS) at0.01% during the growth phase. 10 ml of a 20-hour culture of theorganism grown in nutrient broth plus 250 mg/l phenol plus 0.1 ml of 1%solution of EMS were incubated 30 minutes at 4° C. The culture was spundown, the supernatant decanted, and the cells were resuspended in 10 mlof NS-SSC media and incubated overnight at 4° C. This procedure ofcentrifugation, resuspension in fresh media, and incubation was repeatedtwice. The final culture was streaked onto NS-SSC agar plates and fourcolony types were selected. All four mutants were tested for growth inNS-SSC flasks with increasing amounts of phenol and for growth in NS-SSCmedia where phenol was replaced by the surfactant Igepal CO 660. Oneorganism was selected for its superior utilization of phenol and IgepalCO 660 at 4° C. This organism is herein referred to as the mutantPseudomonas putida CB-173.

The following data demonstrates the ability of Pseudomonas putida CB-173to degrade the surfactant Igepal CO 660 at 4° C. A reverse flow or"upflow" biotower was used. The biotower comprised a cylindrical columnfilled with Pall rings made of plastic resin. The liquid wastewater orsolution to be tested and air were introduced into the bottom with aconcurrent flow of liquid and air up through the column where it drainedoff at the top.

A slime layer of Pseudomonas putida CB-173 was built up on the Pallrings in the biotower by recycling a solution of 2% whey, 0.5% disodiumphosphate and 0.1% (NH₄)₂ SO₄ in water inoculated with the Pseudomonasputida CB-173. After the slime layer had developed, the solution wasreplaced with a test solution of approximately 3500 mg/l Igepal CO 660supplemented with 1 g/l ammonium sulfate and 0.5 g/l disodium phosphate.This was a batch test whereby the solution was not continuously flowedin and decanted off but stayed in the biotower. The Pseudomonas putidaCB-173 was active at 4° C. in reducing the level of Igepal CO 660 asshown by the results in Table 8 below:

                  TABLE 8                                                         ______________________________________                                        RATE OF REDUCTION WAS                                                         180 mg IGEPAL CO 660/hr/ft.sup.3 OF REACTOR                                                            Concentration                                                                 of                                                   Time         Temperature Igepal CO 660                                        hrs.  pH     (°C.)                                                                              (mg/l)    % Reduction                                ______________________________________                                         0    7.75   4           3465      --                                         52    7.80   4           2467      29%                                        74    --     4           2100      40%                                        98    --     4           1617      53%                                        165   7.50   4           1050      70%                                        ______________________________________                                         The method of analysis of Igepal CO 660 used was an analysis for nonionic     described by D. G. Stevenson, "The Adsorptiometric Determination of a         NonIonic Detergent," Dept. of Atomic Energy, Atomic Weapons Research          Establishment, Aldermaston, Berkshire, England (August, 1954).           

When compared to sewage seed from the Roanoke Sewage Treatment Plant,Roanoke, Va., respirometer studies showed the mutant Pseudomonas putidaCB-173 to have much greater activity in nutrient broth substrate at11°-13° C. In addition, the mutant Pseudomonas putida CB-173 was able tobreak down 350 mg/l phenol to less than a detectable level in five daysat this temperature. The results are set forth in Table 9 below.

                  TABLE 9                                                         ______________________________________                                                                             Phenol*                                                                       Concentra-                                                                    tration in                                                           mg O.sub.2 Con-                                                                        Culture of                                     mg O.sub.2 Con-                                                                          mg O.sub.2 Con-                                                                          sumed by CB-173 in                                Hours sumed by   sumed by   CB-173 in                                                                              Nutrient                                 After Sewage Seed                                                                              CB-173 in  Nutrient Broth and                                Inocu-                                                                              in Nutrient                                                                              Nutrient   Broth and                                                                              Phenol                                   lation                                                                              Broth      Broth      Phenol   (mg/l)                                   ______________________________________                                         8    16          18         15      350                                      24    18          97         47      --                                       48    20         393        272      --                                       64    21         443        421      --                                       80    27         475        569      --                                       96    38         503        698      --                                       112   44         529        793      --                                       120   45         532        797      <1                                       ______________________________________                                         *Phenol was assayed by an adaptation of the direct photometric method,        Standard Methods for the Examination of Water and Wastewater, 14th            Edition, 1975, p. 580.                                                   

Respirometer results were taken automatically from stirred one-literreactors in an electrolytic respirometer produced by OceanographyInternational Corp. with temperature control and automatic printout.

A synergistic effect was noted when the combination of a mixedmicroorganism culture containing: Micrococcus species strain, Bacillusspecies strain and two Pseudomonas species strains were used with thePseudomonas putida CB-173. Table 10 below shows the increased activity.Effort was made to have the inoculum in both reactors containapproximately the same number of cells.

                  TABLE 10                                                        ______________________________________                                                                            Phenol Con-                                               mg O.sub.2 Con-     centration                                                sumed by            with Micro-                                               Microorgan-                                                                              Phenol Con-                                                                            organism                                       mg O.sub.2 Con-                                                                          ism Com-   centration                                                                             Combination                                    sumed by   bination   with CB-173                                                                            and CB-173                                Hrs. CB-173     and CB-173 (mg/l)   (mg/l)                                    ______________________________________                                        15   2.7        1.2        --       --                                        25   49         41         --       --                                        35   134        143        --       --                                        50   276        360        300      160                                       70   454        570        130      <1                                        ______________________________________                                         The substrate was nutrient broth, Baltimore Biological Laboratories, with     350 mg/l phenol. Each inoculum was from 24hour shake flask cultures. Tota     amount of each inoculum was the same. Phenol concentration was measured a     50 hours and 70 hours to determine phenol degradation degree. Increased       phenol degradation rates are seen in wastes lower in competitive carbon       sources.                                                                 

The effectiveness of the type strains of Pseudomonas putida versus themutant Pseudomonas putida CB-173 in degrading phenol at varioustemperatures is demonstrated in Table 11 below. Pseudomonas putidaCB-173 grew better at 4° C. than the type strains of Pseudomonas putida,and Pseudomonas putida CB-173 degraded phenol at a much greater ratethan the Pseudomonas putida type strains at all temperatures, especiallyat 4° C.

                  TABLE 11                                                        ______________________________________                                        Temp. Organism                                                                ______________________________________                                                      Phenol Conc. (mg/l)                                                                 Day    Day   Day  Day   Day                                                   3      5     6    9     10                                 4° C.                                                                       P. putida-Biotype A                                                                         280    280   280  275   270                                     P. putida-Biotype B                                                                         280    280   280  265   250                                     P. putida-CB-173                                                                            280    150   10   <1    <1                                              % Transmittance of                                                            Culture to Indicate Growth                                                          Day    Day   Day  Day   Day                                                   3      5     6    9     10                                 4° C.                                                                       P. putida-Biotype A                                                                         100    --    91    56   --                                      P. putida-Biotype B                                                                          95    --    45    52   --                                      P. putida-CB-173                                                                             92    --    41    13   --                                              Phenol Conc. (mg/l)                                                                 Day 1     Day 2                                                               (24 hrs.) (48 hrs.)                                                                             Day 3                                   15° C.                                                                       P. putida-Biotype A                                                                         280       280     270                                           P. putida-Biotype B                                                                         280       280     240                                           P. putida-CB-173                                                                            350        5      <1                                                    % Transmittance of                                                            Culture to Indicate Growth                                                          Day 1       Day 2                                                             (24 hrs.)   (48 hrs.)                                     15° C.                                                                       P. putida-Biotype A                                                                         99          --*                                                 P. putida-Biotype B                                                                         99          --*                                                 P. putida-CB-173                                                                            96          --*                                                         Phenol Conc. (mg/l)                                                                 Day 1       Day 2                                                             (24 hrs.)   (48 hrs.)                                     30° C.                                                                       P. putida-Biotype A                                                                         280         280                                                 P. putida-Biotype B                                                                         280         280                                                 P. putida-CB-173                                                                            280          5                                                          % Transmittance of                                                            Culture to Indicate Growth                                                          Day 1       Day 2                                                             (24 hrs.)   (48 hrs.)                                     30° C.                                                                       P. putida-Biotype A                                                                         53           40                                                 P. putida-Biotype B                                                                         44           40                                                 P. putida-CB-173                                                                            45           41                                           ______________________________________                                         The substrate was nutrient broth, Baltimore Biological Laboratories, with     300 mg/l phenol. Media was prechilled and inoculated with 0.05 ml of a        24hour culture of the indicated microorganism strain. Flasks were shaken      at constant speed and maintained at the temperatures indicated.               *Turbid, indicating abundant growth, measurement unnecessary.            

The Pseudomonas putida CB-173 can be employed alone or in combinationwith other microorganisms conventionally used in microbiologicaltreatment of wastes. This invention also includes the use of anyvariants of Pseudomonas putida CB-173 alone or in combination.

The mutant strain Pseudomonas Putida CB-173 of this invention can becultured in wastewater from any type of industrial, municipal ordomestic source containing a wide variety of phenolics and likematerials either using a batch process, a semi-continuous process or acontinuous process, and such is cultured for a time sufficient todegrade the phenolics and like materials present in the wastewater andremove them or break them down into components capable of being degradedby other organisms normally found in biological wastewater treatmentsystems.

The mutant strain of this invention can be employed in trickling filtersystems, in carbon adsorption systems, in activated sludge treatmentsystems, in outdoor lagoons or pools etc. Basically, all that isnecessary is for the microorganism to be placed in a situation ofcontact with the wastewater. In order to degrade the material present inthe wastewater, the organisms can be cultured under conditions of about1° C. to about 35° C., more generally about 10° C. to about 30° C. Lowtemperature conditions of about 1° to about 10° C., particularly about4° to about 10° C., can also be used with the microorganism strain withunexpected rates of degradation being obtained in comparison with otherorganisms, including other Pseudomonas putida strains, within thistemperature range. Desirably, the pH is maintained in a range of about6.0 to about 8.5, preferably 7.0 to 7.5. Control of the pH can be bymonitoring of the system and an addition of appropriate pH adjustingmaterials to achieve this pH range.

The culturing is conducted basically under aerobic conditions of adissolved oxygen concentration of about 0.5 mg/l or more, preferablyabout 2 mg/l or more. These conditions can be simply achieved in anymanner conventional in the art and appropriate in the treatment systemdesign being employed. For example, air can be bubbled into the system,the system can be agitated, a trickling system can be employed, etc.

The wastewater to be subjected to the process of this invention maycontain sufficient nitrogen and phosphorus for culturing without theneed for any additional source of nitrogen or phosphorus being added.However, in the event the wastewater is deficient in these twocomponents, suitable available nitrogen sources, such as ammonia or anammonium salt, e.g., ammonium sulfate, can be added to achieve anavailable nitrogen content of at least about 5 mg/l or more per 100 mg/lBOD₅. Similarly, phosphorus can be supplemented, if necessary, byaddition of orthophosphates, e.g., sodium phosphate, to achieve aphosphorus level in the wastewater of about 1 mg/l of more per 100 mg/lBOD₅. In general, the treatment is conducted for a sufficient time toachieve the reduction in levels of phenolics and like materials and, ingeneral, about 24 hours to 4 weeks or longer, although this will dependupon the temperature of culturing, the concentration of these materialsin the wastewater and the volume to be treated and other factors, hasbeen found to be suitable.

In the above manner, phenolics and like materials which have beenpreviously considered in the art to be difficultly degradable ornon-biodegradable and which are especially slow or hard to degrade atlow temperatures, as well as other organic compounds which might bepresent in wastewater systems, can be advantageously treated to providetreated wastewater suitable for discharge after any additionalconventional processing such as settling, chlorination, etc., intorivers and streams.

As can be seen from an examination of the examples given herein, themutant strain Pseudomonas putida CB-173 provides advangageous results indegrading phenolics and the like, particularly at low temperatures.

The following description is not to be considered to be limiting, rathermerely exemplary of the types of phenolics to which this invention isapplicable and which can be degraded in accordance with the method ofthis invention. Suitable examples of phenolics and like materials whichcan be degraded include catechols and substituted catechols, phenols andsubstituted phenols including monophenols, ortho-diphenols, etc., anddihydroxyphenylalanines as well as certain phenolic based nonionicmaterials.

In order to further demonstrate the effectiveness of the mutant strainPseudomonas putida CB-173, the following examples are given as exemplaryof the invention but without intending to limit the same. Unlessotherwise indicated herein, all parts, percents, ratios and the like areby weight.

EXAMPLE 1

A field study was conducted on wastewater at the plant of a foodprocessing company in Eastern Pennsylvania during the winter months ofJanuary thrugh March. The culture was added to the wastewater holdinglagoon and the normal parameters of degradation compared to thoseobtained the previous year without the culture containing Pseudomonasputida CB-173.

                  TABLE 12                                                        ______________________________________                                                          First  Second                                                                 Year   Year                                                                   Without                                                                              With                                                                   CB-173 CB-173                                               ______________________________________                                        Ave. Raw BOD        1800     1125                                             Ave. Final BOD      98       12                                               % BOD Reduction     94.6%    98.9%                                            Ave. Raw TSS        1006     1047                                             Ave. Final TSS      65       34                                               % TSS Reduction     93.5%    96.8%                                            Ave. Mixed Liquid Temp. (°C.)                                                              13.5     10.5                                             Lowest Mixed Liquor Temp. (°C.)                                                            9.5      7                                                ______________________________________                                    

EXAMPLE 2

In a further field study presently in progress at a phenol resin plant,it was found that utilization of the Pseudomonas putida CB-173 of thisinvention at low temperatures generally encountered during the wintermonths in that area allowed a reduction in the amount of steam requiredfor lagoon heating to achieve normal lagoon operation, with an estimatedsavings in energy cost thus far in mid January of about $2500 perlagoon.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

What is claimed is:
 1. A biologically pure culture of the microorganismPseudomonas putida CB-173 having the identifying characteristics ofATCC-31800, said microorganism being capable upon culturing inwastewater containing phenolics of utilizing such as an assimilablesource of carbon.